A method for harvesting cells from fermentation broth suitable for automated operation.

Abstract

The present invention discloses a method for recovering bacterial cells from bacterial or fungal fermentation culture broth, the method comprising the steps of: a) mixing a fermentation culture broth obtained from bacterial or fungal fermentation culture with carboxylated chitosan to obtain a mixture; optionally, b) adding a sodium alginate solution to the mixture and mixing uniformly; and c) precipitating to obtain a bacterial cell precipitate or a fermentation culture broth supernatant.

Description

[Technical field]

[0001] The present invention belongs to the technical field of biochemical engineering downstream processing, and in particular relates to a method for harvesting bacterial and fungal cells from fermentation broths that is suitable for automated operation. [Background technology]

[0002] Of the top 30 best-selling drugs in the world in 2021, 24 are biopharmaceuticals. The speed and success of biopharmaceutical research and development is closely related to whether protein samples can be quickly obtained for protein structure, in vitro and in vivo activity studies. Currently, the transient expression process in mammalian cells is the main way to obtain proteins for research. The amount of plasmid DNA required for this process is generally at the milligram to gram level, and depending on the size of the early-stage candidate molecule library, the number of protein samples that need to be produced is generally in the range of tens to thousands, and correspondingly, the amount of plasmid DNA required is also on the order of tens to thousands. In the process of accelerating biopharmaceutical research and development, it is urgent to solve the problem of how to quickly obtain large amounts of plasmid DNA required for protein production.

[0003] Taking bacteria as an example, E. coli is an important host bacterium for genetic engineering in modern biotechnology. It has the advantages of a clear genetic background, fast growth rate, simple genetic manipulation, and easy scale-up of the production process, and is widely used for the production of plasmid DNA, recombinant proteins, enzymes, and other biological products on a laboratory or industrial scale. When using E. coli as a host cell to produce plasmid DNA, E. coli cells are harvested from the culture medium, and the required plasmid DNA is extracted from the E. coli cells. Common methods for harvesting E. coli cells from fermentation broth include high-speed centrifugation and membrane filtration. High-speed centrifugation is usually used to prepare plasmid DNA at the microgram to milligram level in laboratories, and the number of samples processed in a batch is limited and many manual operations are required, making it difficult to expand the throughput and scale; membrane filtration can process a large number of samples at a time, making it suitable for processing a large amount of the same sample, but there are risks such as membrane clogging, damage, and contamination, and the cost of consumables is high, making it difficult to realize multi-sample and high-throughput processing. In addition, both centrifugation and membrane filtration rely on many manual operations, and the investment in facilities and personnel must be doubled as the number of samples processed in a batch increases. Therefore, it is urgent to establish a method for collecting E. coli cells that is suitable for high-throughput and automated processing.

[0004] Chemical flocculation can be used to separate bacterial cells from culture media and is a low-cost alternative to centrifugation and membrane filtration. A published Chinese patent (CN109439584A) shows that chitosan and sodium alginate can concentrate E. coli fermentation broth by flocculation to achieve a bacterial cell enrichment effect. However, this method was only studied for the recovery of E. coli cells, and the impact of the precipitation effect on bacterial cell integrity, product, and more importantly, subsequent plasmid recovery was not studied.

[0005] Chitosan is a linear amino polysaccharide prepared by deacetylation of chitin in hot alkaline solution. Its chemical name is β-(1,4)-2-amino-2-deoxy-D-glucan, its molecular formula is (C6H11NO4)n, and its structural formula is: [ka] .

[0006] Chitosan is insoluble in water and organic solvents, but soluble in dilute acid solutions. Chitosan is insoluble in water, which makes the process of its use complicated, so its application to the aggregation and collection of bacterial cells has not been widely realized.

[0007] Therefore, there is a need in the field to develop a method for collecting bacterial cells that is easy to operate, does not affect the yield of subsequent plasmid production, does not rely on centrifugation or membrane filtration, and can be automated, which is crucial for high-throughput automated preparation of plasmid DNA and rapid production of proteins required for late-stage biopharmaceutical research and development. Summary of the Invention [Problem to be solved by the invention]

[0008] It is therefore an object of the present invention to provide an improved and highly efficient method for harvesting fermented and cultured bacterial and fungal cell mass while maintaining the integrity of their plasmid DNA and RNA, and for extracting protein and nucleic acid products. [Means for solving the problem]

[0009] Thus, the present invention provides a method for recovering bacterial cells from a bacterial or fungal fermentation culture broth, the method comprising the steps of: a) mixing a fermentation culture broth obtained from a bacterial or fungal fermentation culture with a carboxylated chitosan to obtain a mixture; b) adding sodium alginate solution to the mixture and mixing uniformly; and c) Precipitation to obtain a bacterial cell precipitate.

[0010] In another aspect of the present invention, step b) of the above method is omitted, thus in this aspect there is provided a method for harvesting a cell sediment or a fermentation culture broth supernatant from a bacterial or fungal fermentation culture broth, said method comprising the steps of: a) mixing a fermentation culture broth obtained from a bacterial or fungal fermentation culture with a carboxylated chitosan to obtain a mixture; and c) precipitating to obtain a bacterial cell precipitate or a fermentation culture broth supernatant. A preferred embodiment of this aspect also includes a step d) of harvesting the cells after step c).

[0011] In a specific embodiment, the bacterium is selected from Bacillus, Escherichia coli, Lactobacillus, Lactococcus, preferably Escherichia coli or Bacillus subtilis; said fungus is selected from: yeast, preferably baker's yeast, feed yeast, brewer's yeast, Pichia yeast; Candida; mold, more preferably Rhizopus, Aspergillus, most preferably brewer's yeast and Pichia yeast.

[0012] In one preferred embodiment, the bacterium is Escherichia coli and / or the fungus is Pichia yeast.

[0013] In a specific embodiment, the amount of carboxylated chitosan mixed in step a) is 1%-32% by weight of the fermentation culture broth, preferably 1-16% by weight, more preferably 4%-8% by weight, and most preferably 8% by weight.

[0014] In a specific embodiment, the amount of sodium alginate added in step b) is 1%-16% by weight of the fermentation culture broth, preferably 1%-8% by weight, more preferably 4%-8% by weight, and most preferably 8% by weight.

[0015] In another specific embodiment, the amount of carboxylated chitosan and sodium alginate added to the fermentation culture broth is 8% by weight of the fermentation culture broth, respectively, or the amount of carboxylated chitosan added to the fermentation culture broth is 8% by weight of the fermentation medium, and no sodium alginate is added. In one more preferred embodiment, the fermentation culture broth is preferably selected from E. coli fermentation culture broth and Pichia yeast fermentation culture broth.

[0016] In another preferred embodiment, the mass ratio of the added carboxylated chitosan to sodium alginate is 1:4-4:1, preferably 1:2-2:1, more preferably 1:1.

[0017] In a specific embodiment, the carboxylated chitosan is a chitosan in which the amino group (-NH2), or the hydroxyl group (-OH), or the amino group (-NH2) and the hydroxyl group (-OH) are modified to a carboxylated group, wherein the carboxylated group is a C1-C6 linear or branched hydrocarbon group substituted with 1-3 carboxyl groups, preferably containing 3 carboxyl groups, more preferably containing 2 carboxyl groups, and most preferably containing 1 carboxyl group; the linear or branched hydrocarbon group preferably has 1-6 carbon atoms.

[0018] In another specific embodiment, the E. coli strains include E. coli TOP10, E. coli JM109, E. coli HB101, E. coli DH5α, E. coli BL21(DE3), and other derivatives of E. coli K and B. In a preferred embodiment, the yeast strains include Saccharomyces cerevisiae, Pichia pastoris.

[0019] In another specific embodiment, the recovery in step d) is performed by pouring off the medium or pouring off the medium using an automated robotic arm to obtain bacterial cells, disrupting the bacterial cells using a physical or chemical method to release the DNA or RNA within the bacterial cells, adsorbing the DNA or RNA with magnetic beads, washing the DNA or RNA with a washing solution, and purifying the same; preferably by pouring off, more preferably by manually pouring off the medium or pouring off the medium with an automated robotic arm.

[0020] In a specific embodiment of this aspect, after the cells are obtained, the protein may be extracted from the cells.

[0021] In yet another preferred embodiment, the supernatant of the medium collected in step d) is obtained by pouring off the medium or pouring off the medium by an automated robotic arm, followed by purification of DNA, proteins or small molecule metabolites in the supernatant.

[0022] In yet another embodiment, the E. coli fermentation medium is selected from LB broth medium and the yeast fermentation medium is selected from YPD medium.

[0023] Thus, the advantages of the method for recovering bacterial and fungal fermentation culture cells of the present invention are: 1. The use of water-soluble carboxylated chitosan and sodium alginate can effectively sediment the bacterial cells in the fermentation broth, which is simple to operate, low cost, and does not require centrifugation or membrane filtration. 2. The fermentation broth is treated with carboxylated chitosan and sodium alginate, and then the fermentation supernatant is poured off to obtain the precipitated cells. The process can be automated using a robotic arm. (3) The cells obtained by sedimentation are relatively dispersed and do not aggregate to form flocs, which is useful for cell lysis and subsequent extraction of intracellular plasmid DNA, RNA, and proteins. (4) The fermentation broth can also be treated with carboxylated chitosan and sodium alginate, and then the fermentation supernatant can be directly separated by pouring off, which can be used in subsequent biochemical separation processes to obtain the desired products (including, but not limited to, biological metabolic products such as proteins, alcohols, acids, esters, ketones, etc.).

[0024] Additional features and advantages of various embodiments will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by the practice of various embodiments. The objectives and other advantages of the various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.

[0025] Unless otherwise specified, the reagents, cells, and equipment used in the present invention are generally commercially available and publicly available. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows the agarose gel electrophoresis patterns of plasmid DNA extracted from the cells recovered by centrifugation (lane 1) and plasmid DNA extracted from the cells recovered with carboxylated chitosan (lane 2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Reference will now be made in detail to some embodiments of the invention described in the following examples. It will be understood, however, that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all permutations, modifications, and equivalents encompassed within the invention as defined by the appended claims.

[0028] The carboxylated chitosan used in the specific examples of the present specification is a chitosan in which the amino group (-NH2), or the hydroxyl group (-OH), or the amino group (-NH2) and the hydroxyl group (-OH) are modified to a carboxylated group, where the carboxylated group is a C1-C6 linear or branched hydrocarbon group substituted with 1-3 carboxyl groups, preferably containing 3 carboxyl groups, more preferably containing 2 carboxyl groups, and most preferably containing 1 carboxyl group; the linear or branched hydrocarbon group preferably has 1-6 carbon atoms. The carboxylated chitosan has excellent water solubility. Carboxylated chitosans currently available on the market include, for example, carboxylated chitosan manufactured by Aladdin Reagents, which can be prepared according to the methods described in documents such as "Synthesis of carboxylated chitosan and its adsorption properties for cadmium (II), lead (II) and copper (II) from aqueous solutions" (KL Lv, YL Du, CM Wang;. Water Sci Technol 1 July 2009; 60 (2):467-474. doi:https: / / doi.org / 10.2166 / wst.2009.369).

[0029] The bacterial and fungal fermentation cell cultures described herein include techniques in the art for culturing bacterial and fungal cells under appropriate conditions using a fermentation medium as a medium to amplify and produce a desired product of interest. The fermentation cultures herein may be at a laboratory level or at an industrial production level.

[0030] The term "bacteria" as used herein includes, but is not limited to, gram-positive and gram-negative bacteria, particularly bacteria commonly used in fermentation cultures, such as Bacillus, Escherichia coli, Lactobacillus, Lactococcus, etc., preferably Escherichia coli and Bacillus subtilis. The term "fungi" as used herein includes, but is not limited to, eukaryotic spore-forming microorganisms, such as yeast, preferably baker's yeast, feed yeast, brewer's yeast, Pichia yeast, etc.; Candida; molds, such as Rhizopus, Aspergillus, etc., preferably brewer's yeast and Pichia yeast. EXAMPLES

[0031] Example 1 Extraction and analysis of E. coli cells

[0032] 1. Reagents LB broth medium (Sangon Biotechnology, A507002): 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract; Chitosan solution: 1.5% chitosan acetate solution was prepared by dissolving 1.5 g of chitosan (Aladdin Reagent, C105799) in 100 mL of 1% acetic acid solution (FISHER, A35-500). Carboxylated chitosan solution: 1.5 g of carboxylated chitosan (Aladdin Reagent, C105800) was dissolved in 100 mL of ultrapure water to prepare a 1.5% aqueous solution of carboxylated chitosan. Sodium alginate solution: 1.0 g of sodium alginate (Aladdin Reagent, S100127) was dissolved in 100 mL of ultrapure water to prepare a 1% sodium alginate aqueous solution. Bacterial resuspension: 50mM glucose (Hutest, 10010518), 25mM Tris-HCl (pH8.0) (Hutest, 30188336), 10mM EDTA (pH8.0) (Hutest, 10009717). Dissolution solution: 0.2 mol / L NaOH (Hu Test, 10019718), 1% (m / V) SDS (Hu Test, 30166428). Neutralization solution (100 mL): 60 mL 5 M potassium acetate (Fujitsu, 30154518), 11.5 mL glacial acetic acid (FISHER, A35-500), 28.5 mL distilled water. RNase A: 10 mg / mL (Yisheng Biotech, 10405ES03). pUC19 Plasmid: Thermo Scientific SD0061 Top 10 E. coli strains: TIANGEN CB104

[0033] The sequence of the pUC19 plasmid is as follows: 1. pUC19 DNA sequence >pUC19 (SEQ ID NO: 1)

[0034] 2. Method: 2.1 Carboxylated chitosan precipitation treatment of small-scale E. coli fermentation broth Top10 E. coli strain containing pUC19 plasmid was cultured overnight in LB medium to obtain E. coli fermentation broth, and samples were taken to measure OD600. Six 1 mL samples of the fermentation broth were taken for carboxylated chitosan precipitation experiments. Different amounts of carboxylated chitosan solution were added to 1 mL of fermentation broth and mixed by vortexing; then, corresponding amounts of sodium alginate solution were added, mixed by vortexing, and left for 30 min. Another 1 ml of fermentation broth was taken as blank control. The OD600 of the control group and the carboxylated chitosan treatment group were detected. The amounts of carboxylated chitosan and sodium alginate added were shown in Table 1. Based on the OD600 before and after precipitation, the sedimentation rate was calculated; sedimentation rate = (OD600 of original sample - OD600 of sample after precipitation) / OD600 of original sample.

[0035] 2.2 Effect of carboxylated chitosan precipitation treatment of small samples on plasmid DNA extraction Different processing methods were applied to the E. coli bacteria cells of the control group samples and experimental groups 1, 2, and 3 samples prepared by method 2.1. The specific process is as follows: (1) control group: the bacteria liquid was centrifuged at 3500g for 1 minute, and the fermentation supernatant was removed to obtain the bacteria cells; (2) experimental group: the supernatant of the settled fermentation broth was sucked up with a pipette to obtain the bacteria cells that settled at the bottom of the centrifuge tube. The bacteria cells were resuspended with 250μL of the bacteria cell resuspension and 1μL of RNase A, and 250μL of the lysis solution was added and gently inverted to mix uniformly to dissolve the bacteria cells, and 350μL of the neutralization solution was added and gently inverted to mix uniformly. After centrifugation at 12,000g, the supernatant was collected, an appropriate amount of magnetic beads was added, and the mixture was mixed uniformly, and the magnetic beads adsorbed the DNA in the supernatant. Then, a magnet was used to adsorb the magnetic beads, and the supernatant was removed. Next, the magnetic beads were washed twice by adding 70% ethanol, the ethanol was dried, and the magnetic beads were eluted with 50 μL of ultrapure water to obtain plasmid DNA. The plasmid DNA concentration was detected using a spectrophotometer, and the plasmid DNA yield was calculated.

[0036] 2.3 Comparison of the precipitation effects of carboxylated chitosan and chitosan in large-scale E. coli fermentation broth

[0037] 150mL of LB medium was added to two 250mL Erlenmeyer shake flasks, and Top10 E. coli strain containing pUC19 plasmid was cultured overnight to obtain E. coli fermentation broth, and a sample was taken to measure OD600. 8% carboxylated chitosan and 8% sodium alginate were added to the carboxylated chitosan precipitation group as the optimal flocculant usage amount in laboratory-scale tests; 4% chitosan and 2% sodium alginate were added to the chitosan precipitation group as the preferred flocculant dosage amount described in the published patent (CN109439584A). The addition process and operation of the two groups of experiments are as follows: Carboxylated chitosan group: 12 ml (addition amount 8%) of carboxylated chitosan solution was added to a shake flask and shaken horizontally manually for 30 seconds, then the same volume of sodium alginate solution (addition amount 8%) was added and shaken horizontally manually for 30 seconds. The flask was left at low temperature for 4 hours to allow the E. coli cells to completely settle, after which the supernatant was collected, the OD600 was measured, and the sedimentation rate was calculated. Chitosan group: 6 ml (addition amount 4%) of chitosan solution was added to a shake flask and mixed quickly and uniformly on a shaker at 120-150 rpm for 2-3 minutes, then 3 ml (addition amount 2%) of sodium alginate solution was added and mixed slowly and uniformly on a shaker at 50-60 rpm for 2-5 minutes. The mixture was left at low temperature for 4 hours to allow the E. coli cells to completely settle, after which the supernatant was collected, the OD600 was measured, and the sedimentation rate was calculated. The supernatant was slowly poured out from the shake flask by manual pouring, and the volume of the remaining bacterial liquid was measured using a graduated cylinder to calculate the removal rate of the culture supernatant: Sedimentation rate = (OD600 of original sample - OD600 of sample after sedimentation) / OD600 of original sample; Removal rate of the culture supernatant = (culture volume - volume of bacterial liquid after sedimentation) / culture volume.

[0038] 2.4 Effect of carboxylated chitosan and chitosan precipitation treatments on plasmid DNA extraction from large-volume samples For the plasmid DNA extraction test, carboxylated chitosan and the concentrated bacterial solution obtained after chitosan precipitation were used, and as a control group, bacterial cells collected by centrifuging 150 mL of E. coli solution cultured overnight at 3500 g for 10 minutes were used. Because the volume of the bacterial solution remaining after chitosan treatment was large, the medium that could not be poured out cleanly was carefully removed using an electric pipette, and the final volume of the concentrated bacterial solution was the same as that of the carboxylated chitosan treatment group (about 10 ml). 10 ml of bacterial cell resuspension was added to the bacterial cells obtained by centrifugation of the control group, and the bacterial cells were completely resuspended without aggregation. Next, 10 μL of RNase A was added to the bacterial solutions of the control group and the experimental group, and the mixture was shaken horizontally for 10 seconds, then 10 mL of dissolving solution was added, and the mixture was shaken horizontally for 30 seconds to dissolve the bacterial cells, and then 12 μL of neutralizing solution was added, and the mixture was shaken horizontally for 30 seconds. The neutralized solution in the shake flask was transferred to a 50 ml centrifuge tube and centrifuged at 3500 g for 10 minutes, the supernatant was collected, an appropriate amount of magnetic beads was added, and the mixture was mixed uniformly, and the magnetic beads adsorbed the DNA in the supernatant. Next, the magnetic beads were adsorbed using a magnet, and the supernatant was removed. After that, 70% ethanol was added, the magnetic beads were washed twice, and the ethanol was thoroughly evaporated, and then the magnetic beads were eluted with 1000 μL of ultrapure water to obtain plasmid DNA. A spectrophotometer was used to detect the plasmid DNA concentration, and the plasmid DNA yield was calculated. Agarose gel electrophoresis was used to observe the bands and supercoil state of the plasmid. For the experimental procedure of agarose gel electrophoresis, refer to "Molecular Cloning: A Laboratory Manual (Fourth Edition), Michael R. Green, Joseph Sambrook, Cold Spring Harbor Laboratory Press, 2012".

[0039] 2.5 Effect of treatment of large-volume samples with carboxylated chitosan alone on bacterial cell precipitation and plasmid DNA extraction 150mL of LB medium was added to two 250mL Erlenmeyer shake flasks, and Top10 E. coli strain containing pUC19 plasmid was cultured overnight to obtain E. coli fermentation broth, and a sample was taken to measure OD600. 8% carboxylated chitosan was added to the experimental group, as the optimal flocculant usage amount in the laboratory-scale test; the same volume of ultrapure water was added to the control group. The addition process and operation of the two groups of experiments are as follows: Experimental group: 12 ml (8% added amount) of carboxylated chitosan solution was added to a shake flask, which was then manually shaken horizontally for 30 seconds and then allowed to stand at low temperature to allow the E. coli cells to completely settle. The supernatant was collected at 4 and 6 hours, the OD600 was measured, and the sedimentation rate was calculated. Control group: 12 ml of ultrapure water was added to a shake flask, which was then manually shaken horizontally for 30 seconds and allowed to stand at low temperature. After 4 and 6 hours, the supernatant was collected, the OD600 was measured, and the sedimentation rate was calculated. The supernatant was slowly poured out from the shake flask by manual pouring, and the volume of the remaining bacterial liquid was measured using a graduated cylinder to calculate the removal rate of the culture supernatant. Sedimentation rate = (OD600 of original sample - OD600 of sample after sedimentation) / OD600 of original sample; Removal rate of the culture supernatant = (culture volume - volume of bacterial liquid after sedimentation) / culture volume

[0040] 3. Experimental Results and Analysis 3.1 Precipitation effect of carboxylated chitosan on small amounts of E. coli fermentation broth As described in Method 2.1, the effect of using different amounts of carboxylated chitosan and sodium alginate on the cell precipitation effect of 1 mL of overnight culture of E. coli is shown in Table 1. Within a certain range, the cell precipitation rate showed an increasing trend as the amount of carboxylated chitosan and sodium alginate increased. When the amounts of carboxylated chitosan and sodium alginate were 8% and 8%, respectively, the best cell precipitation effect reached 93.5%. Further increase in the amount of the two flocculants did not further improve the precipitation effect and tended to decrease. Therefore, in the subsequent studies, 8% carboxylated chitosan and 8% sodium alginate were used as the precipitation conditions. [Table 1]

[0041] 3.2 Effect of precipitation of bacterial cells with carboxylated chitosan and sodium alginate on the extraction of small plasmid samples For the plasmid miniprep test, the precipitated bacterial cells obtained from the carboxylated chitosan and sodium alginate experimental groups 1, 2, and 3 obtained above were selected, and the bacterial cells collected by centrifugation (as in method 2.2) were used as the control, and the results were shown in Table 2. The plasmid yield in the control group was 9.4 μg, and in experimental group 3, precipitating the cells using 8% carboxylated chitosan and sodium alginate obtained the highest plasmid yield of 14.5 μg, which is 1.5 times that of the control group; furthermore, with the increase in the sedimentation rate of the bacterial cells, the plasmid yield showed a linear improvement trend. The A260 / A280 of the plasmid DNA extracted on a small scale in the control group and the experimental group were both 1.8-2.0, without any RNA or protein contamination, and can be used for further research. The plasmid yield collected by sedimentation of the bacterial cells under small-volume conditions was significantly higher than that obtained by centrifugation of the bacterial cells, suggesting that carboxylated chitosan or sodium alginate may have a certain promoting effect on the plasmid yield. [Table 2]

[0042] 3.3 Precipitation effect of carboxylated chitosan and chitosan on large-scale E. coli fermentation broth The optimal conditions for the appropriate carboxylated chitosan-sedimented bacterial cells obtained by small-scale extraction in method 2.1 of this study: 8% carboxylated chitosan and sodium alginate, and the optimal conditions for bacterial cell precipitation by chitosan and sodium alginate reported in a published patent application (CN109439584A): 4% added chitosan and 2% added sodium alginate were adopted, and a precipitation experiment of 150 mL of E. coli bacterial cells cultured overnight was carried out, and the results are shown in Table 3. As can be seen from Table 3, when the OD600 of the original fermentation broth was close, the precipitation rate of bacterial cells by chitosan was slightly higher than that of bacterial cells by carboxylated chitosan, which were 98.4% and 92.7%, respectively. However, when the culture supernatant was directly poured out and removed, the medium removal rate of the carboxylated chitosan group was much higher than that of the chitosan group, which were 93% and 62%, respectively. After removing the culture supernatant by direct pouring, the remaining bacterial liquid volume in the carboxylated chitosan treatment group was only 10.5 ml. Due to the high medium removal rate after sedimentation, the samples after carboxylated chitosan sedimentation can be automatically processed in batches using an automated robot arm, which contributes to the system integration of the entire downstream automatic operation after bacterial cell sedimentation. [Table 3]

[0043] 3.4 Effect of carboxylated chitosan and chitosan precipitation treatments on plasmid DNA extraction from large-volume samples The yield of plasmid DNA prepared by the alkaline lysis method from the precipitated E. coli cell suspension obtained in 3.3 was examined. Plasmid DNA was prepared as a control using cells collected by centrifugation. Since the volume of the cell suspension after the precipitation treatment of the chitosan group was large, the remaining medium was manually removed using a pipette to reduce the final volume of the cell suspension to about 10 mL. Next, 10 μL of RNase A was added to the cell suspension, and after shaking horizontally to mix uniformly, an alkaline lysis solution was added to dissolve the cell suspension. Plasmid DNA was then extracted according to the method described in 2.4, and the yield of plasmid DNA obtained by extraction is shown in Table 4. [Table 4]

[0044] From the extraction results in Table 4, it can be seen that the plasmid yield obtained by plasmid extraction of the bacterial cells collected by carboxylated chitosan precipitation and those collected by centrifugation are not very different, being 330.5 μg and 324.5 μg, respectively. Sedimentation with chitosan significantly reduced the efficiency of plasmid extraction of the bacterial cells, with a plasmid yield of only 37.2 μg, which is about 11% of that in the carboxylated chitosan-treated group. During the lysis treatment of the precipitated bacterial cells, visual observation showed that chitosan and sodium alginate caused E. coli to form large flocculent bacterial clumps, which were difficult to disperse evenly, and further caused insufficient dissolution during alkaline lysis. The E. coli obtained by precipitation with carboxylated chitosan and sodium alginate has a delicate appearance and can be quickly and evenly dispersed after manual shaking, without affecting the subsequent alkaline lysis (results not shown). Therefore, cell precipitation with chitosan and sodium alginate is suitable for removing the cells in the fermentation broth and collecting the fermentation supernatant, while cell precipitation with carboxylated chitosan and sodium alginate can be used not only to collect the fermentation supernatant but also for subsequent processing of the cells, for example, to extract intracellular biological products (plasmid DNA, RNA, intracellular proteins, etc.).

[0045] Plasmid DNA A 260 / A 280 The test results (Table 2) and the results of the plasmid supercoil ratio shown by agarose gel electrophoresis (Figure 1) also indicate that harvesting the bacterial cells using the carboxylated chitosan method does not affect the quality of the extracted plasmid DNA.

[0046] 3.5 Effect of treating large-volume samples with carboxylated chitosan alone on bacterial precipitation and plasmid DNA extraction The effect of using carboxylated chitosan alone on the precipitation of E. coli cells and the yield of plasmid DNA extraction was investigated. A sample without carboxylated chitosan was used as a control for precipitation efficiency and plasmid yield. The results are shown in Table 5. [Table 5]

[0047] As can be seen from Table 5, the bacterial cell sedimentation rate of the experimental group after 4 hours of sedimentation reached 80.5%, which was lower than the bacterial cell sedimentation rate of 92.7% of carboxylated chitosan and sodium alginate after 4 hours of sedimentation (Table 3). However, when 8% carboxylated chitosan is used alone after 6 hours of sedimentation, the same bacterial cell sedimentation effect (95.2%) can be achieved as when carboxylated chitosan and sodium alginate are combined for 4 hours of sedimentation. Although the sedimentation time when carboxylated chitosan is used alone is 2 hours longer than when two kinds of flocculants are used, the process of adding reagents is simplified, and the risk of inaccurate liquid addition and pipeline blockage caused by the high viscosity of sodium alginate during automatic operation is also avoided, bringing greater application value.

[0048] In addition, when the bacterial liquid volume or batch size of E. coli to be treated needs to be increased, both the centrifugation method and the membrane filtration method have problems such as a limited number of batch processing samples, many manual operations, difficulty in automation, and the need to increase the number of centrifuges and membrane filtration devices accordingly, which increases costs. The sedimentation method of the present invention requires only the addition of one or two types of flocculants, and allows the separation of the bacterial cells and the fermentation medium by gravity sedimentation under low temperature conditions, is simple, low-cost, and can easily be automated and scaled up for batch processing, which is superior to the centrifugation method and the membrane filtration method.

[0049] 4. Summary of results: As mentioned above, the present inventor unexpectedly discovered that the use of water-soluble carboxylated chitosan and sodium alginate, or the use of carboxylated chitosan alone, can avoid centrifugation or membrane filtration, is easy to operate, and effectively achieves the sedimentation of bacterial cells in E. coli fermentation broth. After treating the E. coli fermentation broth with a flocculant, the fermentation supernatant can be removed by pouring out to obtain the precipitated bacterial cells. The process can be automated using a robot arm. The bacterial cells precipitated by the precipitation method of the present invention are relatively dispersed and do not aggregate to form flocs, which is conducive to the lysis of the bacterial cells and the subsequent extraction of intracellular plasmid DNA, RNA, and proteins.

[0050] It will be apparent to those skilled in the art that various modifications and variations can be made to the various embodiments described herein without departing from the spirit or scope of the present disclosure, and therefore, the various embodiments are intended to cover other modifications and variations of the various embodiments that are within the scope of the present teachings. EXAMPLES

[0051] Example 2 Sedimentation of Pichia yeast cells

[0052] 1. Reagents YPD medium: 1% yeast extract (Thermo Scientific, LP0021B), 2% peptone (Thermo Scientific, LP0042B), 2% dextran (Sinopharm Reagent, 63005518). Carboxylated chitosan solution: 1.5 g of carboxylated chitosan (Aladdin Reagent, C105800) was dissolved in 100 mL of ultrapure water to prepare a 1.5% aqueous solution of carboxylated chitosan.

[0053] 2. Method Pichia pastoris strain GS115 was cultured in 4 ml of YPD medium to obtain Pichia pastoris GS115 fermentation broth, and samples were taken to measure OD600. Various amounts of carboxylated chitosan solution were added to the fermentation broth, mixed by vortexing, and left at 4°C. Fermentation broth without added carboxylated chitosan served as blank control. OD600 of the control group and carboxylated chitosan-treated group were detected every hour. Based on the OD600 before and after sedimentation, the sedimentation rate was calculated; sedimentation rate = (OD600 of original sample - OD600 of sample after sedimentation) / OD600 of original sample.

[0054] 3.Results As described in the Methods section, the effect of different amounts of carboxylated chitosan on the cell sedimentation efficiency of 4 mL of Pichia pastoris solution is shown in Table 6. [Table 6]

[0055] Within a certain range, as the amount of carboxylated chitosan added increased, the bacterial sedimentation rate showed an increasing trend. When the amount of carboxylated chitosan added was 8%, the best bacterial sedimentation effect reached 94.2%. Further increasing the amount of carboxylated chitosan added did not further improve the sedimentation effect.

[0056] This embodiment shows that carboxylated chitosan has a good effect on cell precipitation of Pichia pastoris, and can be used to harvest Pichia pastoris cells or to collect the culture supernatant after precipitation, for subsequent protein purification and other uses.

Claims

1. A method for recovering bacterial cells from a bacterial or fungal fermentation culture broth, comprising the steps of: a) mixing a fermentation culture broth obtained from a bacterial or fungal fermentation culture with a carboxylated chitosan to obtain a mixture; b) adding sodium alginate solution to the mixture and mixing uniformly; and c) precipitating to obtain a cell pellet or a fermentation culture broth supernatant.

2. 2. The method of claim 1, wherein step b) is omitted.

3. 3. The method according to claim 1 or 2, further comprising a step d) after step c) of harvesting the bacterial cells or of recovering the supernatant of the fermentation culture broth.

4. The method according to claim 1 or 2, characterized in that the bacterium is selected from Bacillus, Escherichia coli, Lactobacillus, Lactococcus, preferably Escherichia coli or Bacillus subtilis; the fungus is selected from the following: yeast, preferably baker's yeast, feed yeast, brewer's yeast, Pichia yeast; Candida; mold, more preferably Rhizopus, Aspergillus, most preferably brewer's yeast and Pichia yeast.

5. 3. The method according to claim 1 or 2, characterized in that the amount of carboxylated chitosan added is 1%-32% by weight, preferably 1-16% by weight, more preferably 4%-8% by weight, most preferably 8% by weight of the fermentation culture broth.

5. 2. The method according to claim 1, characterized in that the amount of sodium alginate mixed in step b) is 0%-16% by weight of the fermentation culture broth, preferably 0%-8% by weight, more preferably 0% or 8% by weight.

6. 2. The method according to claim 1, wherein the carboxylated chitosan and sodium alginate added to the fermentation culture broth are each 8% by weight of the fermentation culture broth, and preferably the fermentation culture broth is Escherichia coli and Pichia yeast fermentation culture broth.

7. 3. The method according to claim 2, characterized in that the carboxylated chitosan added to the fermentation culture broth is 8% by weight of the fermentation culture broth, and preferably the fermentation culture broth is Escherichia coli and Pichia yeast fermentation culture broth.

8. The carboxylated chitosan has an amino group (-NH ２ ), or a hydroxyl group (-OH), or an amino group (-NH ２ 3. The method according to claim 1 or 2, characterized in that the chitosan is a chitosan in which the hydroxyl groups (-OH) and hydroxyl groups (-OH) are modified to carboxylated groups, wherein the carboxylated groups are C1-C6 linear or branched hydrocarbon groups substituted with 1-3 carboxyl groups, preferably containing 3 carboxyl groups, more preferably containing 2 carboxyl groups, and most preferably containing 1 carboxyl group; the linear or branched hydrocarbon group preferably has 1-6 carbon atoms.

9. The E. coli strain is E. coli. coli TOP10, E. coli JM109, E. coli HB101, E. coli DH5α, E. coli BL21 (DE3) and E. coli BL21 (DE3). coli DH10B, E. coli JM110, E. coli MC1061, E. coli MG1655, E. coli Stbl2, E. coli Stbl3, E. coli T-Fast, E. coli XL1 Blue, E.

5. The method of claim 4, further comprising the step of: culturing the yeast strains of E. coli K and B, including E. coli Rosetta 2 (DE3), or the yeast strains include Saccharomyces cerevisiae and Pichia pastoris.

10. The method according to claim 3, characterized in that the recovery in step d) comprises pouring off the culture medium or pouring off the culture medium using an automated robotic arm to obtain bacterial cells or culture supernatant, adsorbing the DNA / RNA or protein with magnetic beads, washing the DNA / RNA or protein, and preferably pouring off, more preferably pouring off the culture supernatant manually or pouring off the culture supernatant with an automated robotic arm.

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